Herpes simplex virus 2 (HSV-2) causes genital herpes and increases the risk of transmission and infection with HIV. Thus a vaccine for HSV-2 would not only reduce the rate of genital herpes, but also might reduce spread of HIV. Several HSV-2 vaccines have been tested in humans for prevention or reduction of genital herpes disease, but none has been licensed for use in humans. This year we completed a phase 1 clinical trial to test the safety and immunogenicity of a replication-defective HSV-2 vaccine, in humans. This vaccine was developed by Dr. David Knipe at Harvard, and our lab performed much of the preclinical work in mice and guinea pigs. We studied antibody responses in the vaccine recipients and collaborated with David Koelle at the University of Washington to study cellular immune responses to the vaccine. We are currently validating the clinical data and hope to unblind the study later this year to determine safety and immunogenicity of the vaccine. We have also developed a live attenuated candidate HSV vaccine. We postulated that live attenuated HSV-2 would provide more HSV antigens for induction of virus-specific antibodies and cellular immunity than would a subunit HSV vaccine. We previously reported an HSV-2 mutant, HSV2-gD27, in which the nectin-1 binding domain of glycoprotein D (gD2) is altered so that the virus is impaired for infecting neural cells, but not epithelial cells, in vitro and is impaired for infecting dorsal root ganglia in mice. This year we reported that the mutations in HSV2-gD27 were stable when the virus was passaged in cell culture and during acute infection of mice. HSV2-gD27 was attenuated in mice when it was inoculated onto the cornea, intramuscularly (i.m.), intravaginally, and intracranially. Vaccination of mice i.m. with HSV2-gD27 provided better inhibition of challenge virus replication in the vagina than when the virus was used to vaccinate mice intranasally or subcutaneously. Comparison of i.m. vaccinations with HSV2-gD27 versus an HSV glycoprotein subunit vaccine (gD2t) in adjuvant showed that HSV2-gD27 induced larger reductions of challenge virus replication in the vagina and reduced latent viral loads in dorsal root ganglia but induced lower serum neutralizing antibody titers than those obtained with gD2t in adjuvant. Taken together, our data indicate that a live attenuated HSV-2 vaccine impaired for infection of neurons provides better protection from vaginal challenge with HSV-2 than that obtained with a subunit vaccine, despite inducing lower titers of HSV-2 neutralizing antibodies in the serum. We collaborated with Immune Design Corporation to test a novel HSV2 vaccine candidate, G103, consisting of three recombinantly expressed HSV-2 proteins (gD and the UL19 and UL25 gene products) adjuvanted with a TLR4 agonist glucopyranosyl lipid A (GLA) formulated in stable emulsion. Vaccination of mice with G103 elicited antigen-specific binding and neutralizing antibody responses, as well as robust CD4 and CD8 effector and memory T cells. The T cell responses were further boosted by subsequent challenge with live virus. Prophylactic immunization completely protected against lethal intravaginal HSV-2 infection in mice, with only transient replication of virus in the genital mucosa and sterilizing immunity in dorsal root ganglia. The vaccine expanded both CD4 and CD8 T cells induced in mice by previous infection with HSV-2. In the guinea pig model of recurrent HSV-2 infection, therapeutic immunization with G103 was approximately 50% effective in reducing the number of lesions per animal as well as the overall lesions score.